3,5-dinitrosalicylic acid, 5-nitrofurfurylidene hydrazide and compositions containing and methods employing the same for the control of histomoniasis



United States Patent 3,424,845 3,5-DINI'IROSALICYLIC ACID, S-NITROFURFU-RYLIDENE HYDRAZIDE AND COMPOSITIONS CONTAINING AND METHODS EMPLOYING THESAME FOR THE CONTROL OF HISTO- MONIASIS Edward W. Berndt and Robert D.Vatne, Charles City, Iowa, assignors to Salsbury Laboratories, acorporation of Iowa No Drawing. Continuation-impart of applications Ser.No. 525,300, Feb. 7, 1966, and Ser. No. 598,921, Dec. 5, 1966. Thisapplication May 25, 1967, Ser. No. 641,112 US. Cl. 424-232 10 ClaimsInt. Cl. A61k 27/00; C07d /30 ABSTRACT OF THE DISCLOSURE The newchemical compound 3,5-dinitrosalicylic acid, S-nitrofnrfurylidenehydrazide, compositions containing the same and its synergeticprecursors, and treatments applying said compounds and compositions forthe promotion of growth and the control of Histomoniasis in poultry.

CROSS-REFERENCES RELATED This application is a continuation-in-part ofcopending applications Ser. No. 525,300, filed Feb. 7, 1966, and Ser.No. 598,921, filed Dec. 5, 1966.

This invention relates to novel methods and compositions which have anoutstanding potency in promoting the growth and meat producing capacityin poultry and in the prophylactic and curative control of Blackheadinfections. The invention is also directed to a new chemical compoundwhich is the active and responsible factor underlying thesephysiological and chemotherapeutic effects.

Blackhead is a gastrointestinal disease which occurs in turkeys of allages and may also affect a wide variety of other avian species, such aschickens, guineas, quails, pheasants and peafowl. To the veterinarian itis known as Histomoniasis and Infectious Enterohepatitis in view of itsclinical symptoms manifested by an inflamation of the ceca and liver.The etiological factor of the disorder is a microscopic flagellateprotozoan identified as Histomonas meleagridis. The parasite is mostlyharbored by the common poultry cecal Worm Heterakis gallinarum, and itseggs in which it is able to live for long periods of time. It is mainlythis source of infection which is responsible for the transmission ofthe disease.

Birds contract the disease by consuming feed or water contaminated withthe droppings containing the infections organism, or by swallowing cecalworms or their eggs harboring the parasite. Clinical manifestations ofthe disorder are a lowered head, drooping wings and tails, drowsiness,ruflled feathers, dullness, loss of appetite, a persisting yellowish orsulfur-colored diarrhea and a slight depression of temperature. As arule, young poults are susceptible to a rapid onset and short course ofthe disease, and succumb soon after the appearance of the firstsymptoms. Adult birds are usually sick for several days before they dieand show excessive wasting of flesh. A postmortem examination exposesmultiple ulcerations and lesions of the cecal wall, formingyellowish-green cores in the ceca. It has a disagreeable, putrid odorwhich is the result of tissue destruction. The lesions of the liverconsist of irregular, reddened, or gray spots to large necrotic areas.In advanced cases, the peritoneum and mesenteric tissues becomeinvolved.

The prognosis of the disease, especially in turkeys, is mostunfavorable. Mortality is high and may attain a rate of of the flock.The heaviest losses occur during the first three months of life, but arenot limited to that age. Very frequently an outbreak is observed duringthe breeding season. In some regions, marked particularly by heavyrainfalls and wet growing seasons, such as the Midwest and East, theepizootic damage has been so severe that it once compelled a temporaryabandonment of this branch of poultry husbandry.

We have discovered that a new type of heterocyclic compounds possesses aspecific and potent activity against the pathogenic factor of Blackheaddisease. The compound is a derivative of S-nitrofurfural in which thecarbonyl oxygen of the S-nitrofuraldehyde is replaced by 3,5dinitrosalicyl hydrazide so as to form 3,5 -dinitrosalicylic acid,S-nitrofurfurylidene hydrazide as represented by the followingconfiguration OzN For more convenient reference the compound willhereinafter also be referred to as NFSH. It is a yellow solid,crystalline and odorless substance and has a melting point of 227 to 229C.

NFSH may be prepared by reacting 3,5-dinitrosalicylic acid hydrazidewith S-nitrofurfural compounds whose substituent groups in the2-position have reactive capability when contacted with a hydrazide toform a hydrazone derivative having the general structure Moreparticularly, it may be synthesized by interaction of3,5-dinitrosa1icylic acid hydrazide with S-nitrofurfural or its loweracylal or acetal derivatives.

According to a preferred form of this invention our new compound isproduced by the action of S-nitrofurfurylidene diacetate with3,5-dinitrosalicylic acid hydrazide as outlined in the followingequation:

The starting material S-nitrofurfurylidene diacetate is i EXAMPLE 13,5-dinitrosalicylic acid, S-nitrofurfurylidene hydrazide Seventeengrams (0.07 mole) of 3,5-dinitrosalicylic acid hydrazide were suspendedin a mixture of 200 ml. of alcohol, 100 ml. of water, and 10 ml. ofsulfuric acid. Thereupon 17 grams (0.07 mole) of S-nitrofurfurylidenediacetate were added to the suspension which was then heated just belowthe boiling point for one-half hour. The mixture was cooled and filteredand the resulting condensate was washed with aqueous denatured ethanoland subsequently with water. The material was dried at 110 C. and thefinal product was obtained in yield of 23.5 grams (88.7%). Afterrecrystallization from acetic acid, its melting point was determined at227-229 C. The substance on analysis was found to contain:

Carbon (calculated value 39.46) 39.10 Hydrogen (calculated value 1.93)2.05 Nitrogen (calculated value 19.18) 19.40

NFSH is soluble in dimethylsulfoxide to the extent of about 6 g./100 cc.and 1.5 g./100 cc. in dimethylformamide at room temperature. Itssolubility in other common organic solvents was less than 0.3 g./ 100cc. Its solubility in water is 0.034 g./100 cc. at 24 C.

In exploring the histomonicidal activity of NFSH we have observed thesingular and interesting phenomenon that it performs its functionregardless of whether it is introduced into the metabolic system of theanimal as such in form of its synthetic entity, or is generated thereinby anabolic formation from its precursors. As will be apparent from theabove given configuration, NFSH is composed of two structural moieties,namely ONT-1T4:

the S-nitrofurfurylidene or A-portion, and

NO: OzN

aldehyde or S-nitrofurfurylidenediacetate.

The in vivo formation of NFSH from S-nitrofuraldehyde and3,5-dinitrosalicylic acid hydrazide was proved by the followingexperiment.

EXAMPLE 2 Two five week old turkeys were kept without food overnight.One bird was then given a single oral dose of a mixture of 0.25 g. of5-nitrofuraldehyde plus 0.47 g. of

3,5-dinitrosa1icylic acid hydrazide enclosed in a gelatine capsule. Theother turkey received a double dose of this mixture. Three hoursfollowing the administration of the said precursor compounds fecalspecimens from each bird were assayed by thin-layer chromatography. Acombina tion of chloroform and methanol in the proportion of 2:1 wasused as the developing solvent. The bird receiving the double dose wassacrificed 5 hours after medication and specimens of fluids and solidswere collected from the proventriculus, the ventriculus (gizzard), theduodenum, the ileum and the bile. These specimens, as well as excrementstaken 24 hours after administration from the surviving bird were thenchromatographed. To confirm the identity of the presumed spot, bile andexcretory specimens were chromatographed in two different solventsystems, one a mixture of 99 parts of ethanol and 1 part of ammonia, andthe other equal amounts of chloroform. and acetic acid. The separatedcomponents were rendered visible by spraying the plate with a solutionof 0.10% p-dimethylaminocinnamaldehyde and 20% titanium triohloride.

The chromaographic assay of the excreta which were collected 3 hoursafter the administration of the two aforementioned precursors of NFSHrevealed a spot on the plate whose R, value corresponded to that ofNFSH='0.88 in the CHCI :CH OH (2: 1) solvent system. The response of theexcrements in mixture with synthetic NFSH 0n the same plate wasaccentuated by an enrichment of the presumptive spot, indicating thepresence of NFSH within less than 3 hours.

Chromatographic analysis of specimens from various levels of theintestinal tract also demonstrated the presence of NFSH in the gizzard,ileum and bile.

Chromatograms of bile, gizzard and excretory specimens in other solventsconfirmed the in vivo formation of NFSH. Chromatograms developed 24hours after medication revealed barely identifiable amounts of NFSH.

For the purpose of this disclosure the chemotherapeutic efi'ect of NFSHand its synergetic precursors will be illustrated by their applicationagainst H istomonas meleagridis in turkeys for which they are especiallyadapted. Their remedial action has been demonstrated in terms ofsuppression of clinical symptoms and reduction of mortality rates inartificially infected birds.

In accordance with our invention the active ingredients may beintroduced into the animal organism in any fashion or manner in whichthey are apt to form and/ or build up and maintain an effective bloodortissue-level of NFSH. This can be accomplished either by parenteral ororal dispensation at suitable dosage levels.

Parenteral dispensation is preferably carried out by the peritonealroute in suspension in a convenient, nontoxic suspending medium, such ascarboxymethyl cellulose on the 12th day after artificial inoculation.Oral administration may be either by single dosage enclosed in gelatinecapsules or by incorporation of the active drugs into a solid, nontoxicand ingestible vehicle in which it is uniformly and homogeneouslydispersed. Inactive carriers of ingestible nature are any kind ofvegetable food material such as ground corn, corn meal, dried distillersgrain, citrus meal, ordinary grain, mash, scratch and any other normalor commercial rations. The so-medicated feed rations are placed beforethe birds for consumption ad libitum. The drug may also be used asactive ingredient in liquid compositions which can be convenientlyprepared by means of drinking water in which it is suspended with thehelp of skim milk, edible oils, syrups, wetting agents and emulsifiers.

When parenterally dispensed, as little as 50 mg. of NFSH eifected a 100%survival rate, while at least twice the amount of each precursor wasneeded to bring about a chemotherapeutic response to a concurrentinjection in pari situ.

Oral treatment by single dosage units is likewise applied on the 12thday after infection and requires a minimum of 50 mg. of NFSH, and twicethat amount of each precursor given at the same time.

-In feed the active proportions for the precursor mixture start at theapproximate level of 0.01% for each of the synergetic components. Theminimum remedial doswhile the other infected group remained untreatedand served as controls. Medicated feed nations were placed before theinfected birds for ingestion ad libitum. The birds were held underobservation for an additional seven days after medication to determinethe extent of possible age of NFSH as synthetic material was determinedat 5 relapses. 0.002% by weight of the feed, but concentrations up toArtificial infection was carried out by administration 0.2% and evenhigher may be used for more effective of approximately 1,000 embryonatedeggs of the cecal action, particularly in case of a moderately heavy ex-Worm of chickens, known as Heterakis gallinarum. Durposure of a flock.Preferred proportions for prophylactic ing all the experiments, weightand feed consumption oontrol lie within the approximate range of 0.005to records were kept on each poult under test. It was found 0.01%. thatat the dosage levels dispensed, no detrimental eliect EXAMPLE 3 on thenatural growth or weight increase of the birds was caused by the activeingredient of the compositions. A given number of Broad Breasted Bronzeturkey poults The results of the trials were tabulated in the followw rreared m Mrs-bottom cages enclosed 1n broodmg ing tables. Column 1records the compounds applied. Colrooms. When these po l s We e a t 6Weeks Of g umn 2 shows the number of turkeys employed in each W werePlawd 1H mdlvldllalfiages, havlng test, followed by column 3 listing thedosage levels used. cloth bottoms, Where y l'emalned 2 t0 3 y TlllsColumn 4 records the clinical incidence in terms of the perlod ofisolation allowed the poults to get accustomed number of bird anif tinsym tom of the disease, to their new quarters before the tests werestarted. The These values are translated in column 5 into clinicaleffilot was then divided into 2 groups each of equal n-umcacy expressedin percentages. Column 6 shows the morbers. One of the groups was placedon medication on the tality rates in terms of dead birds which are thenconday of infection and remained on medication for 21 days, verted intosurvival eificacy by percentages in column 7.

TABLE I Single dosage treatment (oral) Compound N0. Dosage, Olin. Clin.Surv.

turkeys mg. incidence eflicacy Mort. rate,

percent 4 Inf. controls 4 0 3 25 5-nitrofuriurylidene diacetate 4 100 40 4 0 3,5-dinitrosalicylic acid hydrazide 4 100 4 0 4 05-uitrofuraldehyde 4 100 4 0 3 25 5-nitrofurturilidene diacetate 4 10.04 0 2 8,5-dinitrosalicyiic acid hydrazide 100 G-nitrofuriuraldehyde 410B 4 0 2 50 3,5-dinitrosalicylic acid hydrazide 100 NFSH 4 50 4 0 0 100NFSH 4 100 4 0 1 NFSH 4 200 4 0 1 75 TABLE II Single dosage treatment(intraperitoneal) Compound No. Dosage, Olin. Olin. Surv.

turkeys mg. incidence efiicacy Mort. rate,

percent 4 Int. controls 4 0 3 25 5-nitroiuriurylidene diecetete 4 4 0 40 3,5-dinltrosalicylic acid hydrazide 4 100 4 0 4 0 G-nitroiuraldehyde 4100 4 0 3 25 fi-nitroiuriurilidene diacetate 4 4 0 0 1003,5-dinitrosa1ieyllc acid hydrazide 100 fi-nitroturaldihyde 4 10B 4 0 050 3,5-dinitrosalicylic acid hydrazide 100 NFSH 4 100 4 0 0 100 NFSH 4200 4 0 0 100 NFSH 4 50 4 0 0 100 TABLE III.-MEDICATED FEED TREATMENTWITH NFSH PRECURSORS No. Dosage, Olin. Clln. Surv. Compound turkeyspercent incidence eificacy Mort. rate,

percent 4 Int. controls 4 4 0 b-nltroiuriurylidene dlacetata 0. 0250 4 04 0 3,5-dlnitrosallcyllc acid hydrazide 4 0.0250 4 0 4 0B-nltro-Z-iuraldehyde 4 0. 0250 4 0 4 0 fi-nitrofuriurilldene diacetate4 0. $250 1 75 0 100 3,6-dinltrosallcyllc acid hydrazide 0.0250

nitrofuraldihyde 4 0. 0350 0 100 0 100 3,5-dlnitrosa1icylic acidhydrazide 0. 0250 3,5-dlnitrosalicyllc acid hydrazide 4 0. 0332 4 0.0166

b nitroiuriurylidcne diacetate 4 0. 0333 4 0.0167

5nitrofuraldehyde 4 0. 0193 3,5-dinitrosa1lcylic acid hydrazide 4 0.0332 fi-nitroiuriurylidene diacetate 0. 0333 3,5-dinitrosalicylic acidhydrazide 4 0. 0166 5-nltroiurfurylidene diacetate 0. 0167 4 0 2 503,5-dlnitrosalicylic acid hydrazide 4 0. 0332 enitroiuraldehyde 0, $93100 0 100 3,5-dinltrosailcylic acid hydrazide 4 0. 0100 0 4 0b-nitrofuraldehyde. 4 0. 0100 0 4 0 B-nitroiuraldehyde 4 0, 01003,5-dinitrosallcyllc acid hydrazide 0.0100 3 1 7 TABLE IV.-MEDICATEDFEED TREATMENT WITH the Poultry industry make it q y desirable to enhanCe SYNTHETIC NFSH the hysiological development and growth of the ammalsDosage Clinical Clinical Mortap survival above and beyond their naturalrate of maturation. In so No. turkeys level, inciefficacy, ity etficacy,promoting the meat producing capac1ty Of his flocks, the percent percentrate percent poultry raiser will be in the position to bl'lllg hlS birdsto 3-- 0 83 s1 market at an earlier date and thereby not nly Increase(0025 6 218 3 the effectiveness of his commercial operat on, but also ii38% g 0 10M achieve substantial savings in nutritlve supplies,especially 105:: 0: 0050 4 301% 8 i881; when the treatmentsimultaneously heightens the feed gg i33 0 100-0 elficiency of thesupplemental basal rations. 11' 010100 1 901 8 $03 We have discoveredthat these valuable results are profg-ggg 8 i888 0 100.0 duced by NFSHas will be demonstrated herein below by 1 010300 0 m 3 $18 a number oftests conducted on turkeys. Conveniently, the 8-8283 3 igg-g 0 100-0mode of administration of the compound likewise con- 41111 11 0 0 100103 188:8 sists in its incorporation into an orally ingestible non-toxic 40.2000 0 100.0 0 100.0 i h as an nimal feed as hereinbefore described-Ini. controls. In the tests tabulated below, 18 newly hatched male andAs will be apparent from the foregoing tables, the efiicacy of thesynthetic drug is about 12 to 22 times superior to that of the precursormixture, especially when the treatment is carried out in form of amedicated diet for a period of 21 days.

The foregoing Table IV shows that all the 81 infected but untreatedcontrols showed severe clinical symptoms several days after inoculationand had succumbed to the disease at the end of the 21-day experimentalperiod. Contrary thereto, test birds receiving a protective NFSH dosageof 0.002% in the feed showed a comparative clinical efiicacy of 65.9%and a survival efiicacy of almost 80%. All the infected poults receivinga medicated ration containing from 0.003% up were increasingly free ofclinical symptoms and attained a survival rate of 100% after 21 days. Norelapses were noted after 7 additional days of observation.

The new compound has also been found to be of beneficial value inproductive poultry management which is geared to the objective ofensuring and maintaining the growth and health of the animals. Whileproper nourishment, enriched with nutritional adjuvants, in conjunctionwith sanitizing measures is vital for rearing a healthy and productiveflock, economic efficiency and profitability in 75 female turkey poultswere treated for 8 weeks with dosages of 50, 200, '600, and 1200 partsper million of NFSH in the feed. Control groups of 18 male and femalepoults each received no dietary treatment. The mean weight gains weredetermined by carefully maintained weight records for each bird and theratios of weight increases were calculated. These figures multiplied byrepresent the percentages of weight gains attained by the treated birdsas compared with the untreated controls. Thus, for example, if the meanweight gain for a group of tested turkeys 'was found to be 4.87 lbs.while the control group showed a value of 4.58 lbs., the percentage ofweight gain expressed as Ratio T C percent is are tabulated the weightgains in lbs., while column 5 translates these values into T C percentratios.

TABLE I Starting Terminal Weight 770, Gone. feed, p.p.m. weight weightgain percent (lbs) (1bs.) (lbs.)

Females:

0 (controls) 0. 13 4. 00 3. 87 100.00 0. 13 4. 3. 97 102. 58 0. 13 4. 204. 07 105.17

As can be seen from the foregoing table, the growth promoting activityof NFHS may rise to a level of l-l18% which is a significant increaseover the fleshing rate of normal physiological development.

Compound NFSH and its precursors may be dispensed in its medium ofadministration either alone or in combination with other healthpromoting or disease control agents. Such additional ingredients may beselected from the group of vitamins, antibiotics and other growthstimulants. Especially, 3 nitro-4-hydroxyphenylarsonic acid, arsanilicacid, and a variety of known coccidiostats or Blackhead remedies whichwill be readily apparent to the veterinarian may be used.

For practical purposes of marketing and to facilitate the handling ofthe minute amounts of our novel remedies to be incorporated into theultimate feed rations, it is most desirable and advantageous to preparea standard concentrate of active ingredients which may be as high as 95%by weight of the composition. To that effect, nontoxic, inert materialssuch as fullers earth, talcum, bentonitc, ground oyster shells,limestone, divers clays, soybean meal, wheat middlings, corn germ mealand corn meal may be selected as the carrier medium. Such stockconcentrates are specifically made and adapted for use in dilutions withan alimentary dispensing vehicle or element of sustenance so as tocompound the medicated rations at their desired dosage levels withutmost convenience. The availability of such commercial concentrates orpremixes, therefore, is indispensable to the feed manufacturer andpoultry raiser who ordinarily uses a standard weight package of premixfor each 1,000 lbs. or one ton of commercial feed to produce themedicated compositions.

10 What we claim is: 1. 3,5-dinitrosalicylic acid, S-nitrofurfurylidenehydrazide represented by the formula OzN N 01 2. A veterinarycomposition useful in the control of Histomoniasis in poultry containinga nontoxic, orally ingestible vehicle and an eifective but nontoxicamount of the compound defined in claim 1.

3. A veterinary composition effective in the control of Histomoniasis inpoultry containing a nontoxic, orally ingestible vehicle and from 0.002%to 0.2% by weight of the compound defined in claim 1.

4. A veterinary composition eflective in the control of Histomoniasiscontaining a turkey feed comprising from 0.002 to 0.2% by weight of thecompound defined in claim 1.

5. A veterinary composition effective in promoting the meat producingcapacity of poultry containing from 0.005% to 0.12% by weight of thecompound defined in claim 1 and a poultry feed.

6. A veterinary composition effective in the control of Histomoniasis inpoultry consisting of a gelatine capsule enclosing an amount of 50 mg.to 200 mg. of the compound defined in claim 1.

7. A veterinary composition effective in the control of Histomoniasis inpoultry consisting of an injectible nontoxic suspending mediumcontaining an amount of 50 mg. to 200 mg. of the compound defined inclaim 1.

8. A method of controlling Histomoniasis in poultry comprising orallyadministering to poultry afilicted with Histomoniasis an etfective, butnontoxic amount of the compound defined in claim 1.

9. A method of controlling Histomoniasis in poultry comprisingparenterally administering to poultry afi'licted with Histomoniasis aneflective, but nontoxic amount of the compound defined in claim 1.

10. A composition suitable for addition to a poultry foodstuffcomprising a non-toxic inert solid carrier material and at least 5% byweight of the compound defined in claim 1.

References Cited UNITED STATES PATENTS 3,330,724 7/1967 Van Essen et a1.167-531 ALBERT T. MEYERS, Primary Examiner.

R. BARRESE, Assistant Examiner.

U.S. Cl. X.R.

